CN103421793B - Cloning and application of swine liver specificity expression gene TTR promoter - Google Patents

Abstract

The invention belongs to the technical field of animal gene engineering, in particular to cloning and activity analysis of a swine liver specificity expression gene TTR promoter with the length of 2205 bp. A 2205 bp-long regulatory sequence at the upstream of a translation initiation codon ATG of the swine liver specificity expression gene TTR promoter is cloned from a swine genome, and the nucleotide sequence is shown as the SEQ ID NO: 3; the result shows that the TTR promoter with the length of 2205 bp has independent activity and liver tissue specificity with the nucleotide sequence shown as the SEQ ID NO: 3. The invention further discloses a preparation method of the promoter fragment, a dual-luciferase activity detection system and the application of the RT-PCR method for the activity analysis of the promoter.

Description

The clone of swine liver specificity expression gene TTR promoter and application

Technical field

The invention belongs to animal gene engineering technology field, be specifically related to isolation identification and the functional verification of Animal muscles different expression gene TTR promoter region.

Background technology

The expression of higher organism gene is subject to the finely regulating of intraor extracellular environment, thus has strict time and spatial ordering.The expression regulation of gene is a complexity and orderly process, is jointly completed by multistage regulation and control level, this mainly comprise transcribe before, transcribe, transcribe after, translate, translate rear five levels.Wherein the regulation and control of transcriptional level are the links of most critical.Promotor is an important controlling element on transcriptional level, is to be positioned at the DNA sequence dna that structure gene 5 ' holds upstream, can with the expression of numerous transcription factor interaction regulatory genes (military force 1999).Therefore further investigate the structure of promotor, function, binding mode etc. to be conducive to us and better to understand the function of corresponding gene and intergenic interaction.

Promotor sorting technique is more, and the function and effect mode according to promotor can be divided into constitutive promoter, tissue specific promoter and inducible promoter; Core promoter, proximal promoter and Proximal promoter (Wang and Hannenhalli2006 can be divided into according to the structure of promotor and position; Reddy et al2003; Aneja et al2008); Structure according to core promoter can be divided into again concentrated promotor and decentralized promotor.Although the classification of promotor is varied, for eukaryote, rna plymerase ii type promotor plays Main Function (Sandelin et al2007).Often only express at some specific organ or tissue position at the gene of tissue-specific promoter's regulation and control, and show the feature of Growth adjustment.The tissue-specific promoter possessing high transcriptional activity is the desirable promotor in genetically engineered, this kind of promotor efficiently can start exogenous gene expression, foreign gene can be made again to express in specific target cell or target tissue, and the CMV of this utilization more extensive than present stage and SV40 promotor have more research and practical value.

Tissue-specific promoter is usually based on specific tissue cellularity with chemistry, physical signalling, target location can be carried out to foreign gene, gene is fixed on a certain tissue or cells, decrease the loss of human body energy and material and the impact on organism metabolism activity, can play again the directed effect improving a certain tissue characteristics, this prospect in human disease treatment is especially considerable simultaneously.Secondly, in transgenic animal breeding research, expressed in target tissue by tissue-specific promoter's regulation and control external source goal gene, effectively can improve some proterties of animal, as intramuscular fat; Or the Digestive tract improving animal is to reduce the nitrogen and phosphorus content etc. in movement.Given this, tissue-specific promoter is more and more subject to the favor of investigator in genetically engineered and transgenic breeding.

TTR gene, be translated as transthyretin (transthyretin, TTR) prealbumin (prealbumin is also called, PA), VA operates albumen, and molecular mass is about 54ku, primarily of liver cell synthesis (Zeledon et al2006), transthyretin 90% is produced by liver, and be then secreted into human cerebrospinal fluid and blood (Chen et al2005), choroid plexus and retina also produce the small segment of this protein.

Up to the present, the relevant report of the promoter function of research pork liver visceral muscle different expression gene TTR is not seen.So applicant has carried out the functional study of promotor to this gene, to this promotor can be utilized better.

Summary of the invention

The object of the invention is to liver specific expression's gene TTR promoter region of separating clone pig, and functional verification is carried out to it, to obtaining possessing higher startup activity and keeping the specific regulating and controlling sequence of muscle tissue, for animal genetic engineering provides available tissue-specific promoter of this gene.

The present invention is separated and identifies TTR gene from the genome of Large White has the specific promotor of liver organization.With the poba gene group DNA of Large White for template amplification obtains the promoter fragment of 2185bp length, this segment composition reporter gene firefly luciferase gene (being called for short LUC gene) is built pGL3-TTR-pro carrier for expression of eukaryon, by these carriers transfection C2C12 respectively, 3T3-L1 and Hepa1-6 cell, by the relative reactivity of Dual-luciferase reportor systerm analysis report gene Photinus pyralis LUC, thus obtain the maintenance specific promoter region of liver organization, the recombinant vectors of this promotor of further structure and BGL1 gene, BGL1 gene comes from the genome of saccharomycopsis fibuligera (Saccharomycopsis fibuligera), the expression amount of BGL1 is detected by RT-PCR.

Particularly, the present invention is realized by following technology:

Technological line of the present invention as shown in Figure 1.

Utilize ncbi database (http://www.ncbi.nlm.nih.gov) search to obtain the upstream regulatory sequence of liver specific genes TTR, utilize bioinformatics software forecast analysis core promoter region, cis-trans functional element and the CpG island distribution situations such as TESS, TFSEARCH and MethPrimer.Design 1 pair of primer (its nucleotide sequence is in table 1), from the genome of Large White, pcr amplification obtains liver organization specificity promoter, applicant is by the promotor called after TTR-pro of amplification, and its fragment length is that its nucleotide sequence of 2185bp is as SEQ ID NO:3.Build reporter gene LUC fusion vector, called after pGL3-TTR-pro.Transient transfection C2C12,3T3-L1 and Hepa1-6 cell, Dual-luciferase reportor systerm detects and finds that TTR promotor TTR-pro shows comparatively significantly relative reactivity in Hepa1-6 cell, and its activity is higher than negative control about 15 times.The TTR-pro promotor of result display 2185bp possesses the expression specificity that the function independently starting reporter gene expression also remains liver organization simultaneously.Using this sequence as promotor, build the recombinant expression vector with BGL1 gene, by the carrier called after pTTRS-BGLhis-control built.By pTTRS-BGLhis-control transfection C2C12 cell, RT-PCR detects the expression of BGL1 gene.Found that, TTR-pro can drive the expression of foreign gene BGL1, proves that isolated promotor TTR-pro has both the ability starting luciferase reporter gene and BGL1 gene.

Concrete steps of the present invention are as follows:

By US National biological study institute website NCBI(http: //www.ncbi.nlm.nih.gov/) on search pig TTR gene, this gene order is as shown in SEQ ID NO:1, using this sequence as probe sequence, utilize the method for comparative genomics, by the BLASTn database of this DNA sequence dna input NCBI, search obtains the upstream regulatory sequence (its nucleotide sequence is as shown in SEQ ID NO:2) of this gene, utilize TESS, the bioinformatics softwares such as MethPrimer predict its core promoter region to sequence SEQ ID NO:2, cis-trans functional element and CpG island distribution situation, according to the result design primer of neural network forecast, with Large White (from Wuhan City, Hubei Province Hua Zhong Agriculture University test pig farm) genomic dna for template, pcr amplification obtains promoter region, its nucleotide sequence is as shown in sequence table SEQ ID NO:3.These promotor candidate segment are loaded into pGL3-basic reporter gene LUC upstream multiple clone site (information such as carrier figure and multiple clone site is shown in Fig. 2) place, are assembled into fusion expression vector (Fig. 3).Proceed to C2C12,3T3-L1 and Hepa1-6 cell by liposome-mediated infection protocol (Zhou Xueyan and Guan Wei army 2005) by instantaneous for fusion expression vector, proceed to Renilla luciferase reporter gene pRL-TK is internal reference plasmid simultaneously.The present invention arranges negative control pGL3-basic(Fig. 2), positive control pGL3-control.By Dual-luciferase reportor systerm, quantitative analysis is carried out to reporter gene LUC after transfection 48h, and then investigate the start-up performance of this promotor of checking in different sources cell.Further, the promotor obtained is connected the CDS region of BGL1, build recombinant expression vector, the CDS regional sequence of BGL1 is as shown in SEQ ID NO:4.After recombinant expression vector transfection Hepa1-6 cell 48h, extract cell RNA, the expression amount of BGL1 in Hepa1-6 cell is detected by RT-PCR, prove that the core promoter of liver specificity can not only start reporter gene expression with this, also possess the ability of the BGL1 genetic expression starting external source simultaneously.

The invention has the advantages that:

(1) what the present invention was detailed demonstrates the pig TTR promotor ability that promotor gene is expressed in different sources cell, and the expression regulation for TTR gene brings deeper cognition.

(2) the present invention identifies the function of the promotor startup reporter gene of liver organization specifically expressing and the BGL1 genetic expression of external source, for genetically engineered and molecular breeding provide the promotor resource of new specifically expressing.

More detailed technical scheme is see the content of " embodiment ".

Accompanying drawing explanation

Sequence table SEQ ID NO:1 is the full length sequence of the present invention as the pig liver specific expression gene TTR of probe, and sequence is 9915bp(length is 9.91kb).Pig TTR gene is 397419 in the accession number of Genebank.

Sequence table SEQ ID NO:2 be the present invention from database by probe obtain the upstream regulatory nucleotide sequence of pig liver specific expression gene TTR, length is 2585bp.

Sequence table SEQ ID NO:3 is the upstream regulatory nucleotide sequence of the pig liver specific expression gene TTR that the present invention clones, and length is 2185bp.

Sequence table SEQ ID NO:4 is the BGL1 gene C DS sequence (the Genebank accession number of BGL1 gene is 170807) that the present invention clones, and length is 2654bp.

Sequence table SEQ ID NO:5 is the present invention's TTR gene promoter of cloning and signal peptide sequence, and length is 2220bp.

Fig. 1: Technology Roadmap of the present invention.

Fig. 2: expression vector pGL3-basic structural representation, includes reporter gene luc+ in the below of multiple clone site.

Fig. 3: what build take pGL3-basic as the fusion vector sketch of skeleton.The promoters driven LUC genetic expression of pig liver specific expression gene TTR, is represented by the deleted promoter in figure.Amp is ammonia benzyl resistance screening gene; Luc+ is reporter gene Photinus pyralis LUC; MCS represents multiple clone site; The direction of arrow represents the direction of gene or promoter expression.

Fig. 4: the Mlu I of fusion vector and Xho I double digestion qualification figure.M represents DL marker10000, and the double digestion result of 1 expression fusion vector pGL3-TTR-pro, band larger is above carrier ribbon, and lower end is deletion promoters band, and band length is 2205bp.

Fig. 5: by the expression of promoters driven LUC gene in the cell of different sources of liver-specific expression gene TTR.

Fig. 6: carrier pGL3-BGLhis-Control structural representation.

The enzyme of Fig. 7: carrier pGL3-BGLhis-Control cuts qualification figure.M represents DL marker10000, the 1 CDS district representing BGL1 gene

Fig. 8: the structural representation of recombinant expression plasmid pGL3-TTRs-BGLhis-control.

The enzyme of Fig. 9: recombinant expression plasmid pGL3-TTRs-BGLhis-control cuts qualification figure.M represents DL marker10000, and 1 represents TTR-pro fragment.

Figure 10: RT-PCR detects the expression of foreign gene BGL1 in Hepa1-6 cell.

Figure 11: be purchased carrier pGL3-Control Vector structural representation.

Embodiment

Embodiment 1: the acquisition of the promoter fragment of liver specific expression's gene TTR of pig

Ncbi database (http://www.ncbi.nlm.nih.gov/) search the TTR gene of pig DNA sequence dna (GenBank accession number: 397419), search for this gene mRNA 5 ' end upstream 2000bp to First Intron region as regulation and control region.Utilize bioinformatics software prediction cis-trans functional element and the CpG island distribution situations such as TESS, TFSEARCH and MethPrimer.According to the primer of the result of neural network forecast design total length promotor, (primer sequence is as shown in table 1; PCR reaction parameter arranges as shown in table 2).Add upstream primer 5 ' and hold interpolation restriction enzyme site Mlu I(ACGCGT), simultaneously with protection base CG; Downstream primer 5 ' is held and is added Xho I(CTCGAG) restriction enzyme site, with protection base CCG.With the poba gene group DNA of Large White for template, PCR first increases total length promoter sequence TTR-pro, and amplified production to detect and Omega company reclaims after kits reclaims through 1.5% agarose electrophoresis, put to-20 DEG C for subsequent use.Primer sequence, in table 1, has single line place to be restriction enzyme site under base.

Table 1: the design of pig TTR promotor amplimer

Table 2 pcr amplification parameter

Embodiment 2: the promotor transfection carrier of liver specific expression's gene TTR of pig builds.

(1) restriction enzyme Mlu I and Xho I enzyme cut carrier pGL3-basic(carrier purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega company, the information such as carrier and multiple clone site is shown in Fig. 2) and the recovery product of TTR-pro promoter fragment.Enzyme cuts system in table 3

Table 3 enzyme cuts system

Detect enzyme with the agarose gel electrophoresis of 1.5% after 37 DEG C of enzymes cut 4h cut result and reclaim object fragment: pGL3-basic reclaims larger fragment, and TTR-pro reclaims the fragment of corresponding 2205bp.Reclaim test kit with the glue of Omega company to reclaim (by the operation of this test kit specification sheets), be placed in-20 DEG C of Refrigerator stores.

(2) promoter fragment that enzyme cuts back to close is connected to (see figure 3) on pGL3-basic carrier, linked system is in table 4.

Table 4 linked system

16 DEG C of water-baths are spent the night connection, proceeded in bacillus coli DH 5 alpha, screening positive monoclonal containing on the LB flat board of penbritin (Amp), PCR being carried out to recon and enzyme cuts qualification, positive recombinant being chosen and send the prosperous bio tech ltd order-checking of Beijing AudioCodes.For the correct positive colony that checks order in original bacteria liquid: the ratio of culture volume ratio=1:100 carries out enlarged culturing, with the plasmid that the can be used for cell transfecting extraction agent box (D6950-01 in a small amount that OMEGA company produces after 37 DEG C of shaking tables shake 16h, operate by the specification sheets of this test kit) extracting plasmid, called after pGL3-TTR-pro.Identify the plasmid of extracting with Mlu I and Xho I double digestion, it is described above that enzyme cuts system, and now plasmid only need add 1 μ L, and enzyme is cut and be the results are shown in Figure 4, is placed in-20 DEG C of refrigerators for subsequent use.

Embodiment 3: the cell transfecting of recombinant plasmid pGL3-TTR-pro

The present invention does transfection with 24 porocyte culture dish and uses.In order to eliminate the error of experiment, each recombinant vectors all carries out independently testing for 3 times, and 3 repetitions are done in each test.According to Fugene HD(Roche company) specification sheets operation, every hole is in the quality of carrier: the ratio transfection of the volume=0.5 μ g:1.5 μ L of transfection reagent.The acceptor of transfection is the cell of different sources, mainly comprises Mouse Muscle source cell system C2C12(purchased from Chinese Academy of Sciences's cell bank), mouse Preadipocyte In Vitro 3T3-L1(is purchased from Chinese Academy of Sciences's cell bank) and murine hepatocarcinoma cell Hepa1-6(Wuhan Boster Biological Technology Co., Ltd.).The activity of Dual-Luciferase detection system promotor is utilized to analyze after recombinant vectors pGL3-TTR-pro is transfected into cell 48h after collecting cell lysate.Described in being formulated as follows of the key step of cell cultures of the present invention, transfection and various solution:

(1) preparation of main solution

1) cell growth medium (the foetal calf serum substratum FBS of 10% concentration): foetal calf serum (purchased from GIBCO company) is positioned over 4 DEG C of refrigerators and melts, after it melts completely, draw the DMEM/F12 substratum (purchased from HyClone company) that 10mL foetal calf serum moves into 100mL, 0.25 μm of rearmounted 4 DEG C of refrigerator of frit is for subsequent use.

2) cells frozen storing liquid: the volume ratio of dimethyl sulfoxide (DMSO) (DMSO), the low sugar culture-medium of DMEM/F12, foetal calf serum three is 1:4:5, and the frozen storing liquid prepared is frozen in-20 DEG C.

(2) cellar culture of cell

1) change nutrient solution according to the upgrowth situation of cell, within general 1 or 2 day, change once;

2) can go down to posterity time more than cell density to 80%, with the nutrient solution that suction pipe sucking-off is old, with phosphate buffered saline buffer, namely PBS(is purchased from HyClone company) rinse twice.By 0.25% trypsin of 37 DEG C of preheatings purchased from GIBCO company) add culturing bottle, general 50cm ²tissue Culture Flask adds 400 μ L, is paved by pancreatin to cover all cells at the bottom of cell bottle, sucking-off pancreatin after room temperature placement 15s-30s;

3) pat cell bottle sidewall 30s-60s gently, cell is come off from bottle wall, adds a certain amount of fresh 10%FBS nutrient solution in time, the cell being attached to bottle wall is rinsed and stops pancreatin effect;

4) do not rinse the attached cell got off with suction pipe featheriness, make the cell on bottle wall depart from formation cell suspension completely, avoid the generation of bubble as far as possible;

5) blow and beat cell evenly after, the cell suspension of sucking-off 1/2 or 1/3 moves in new cell bottle, puts into 37 DEG C, 5%CO after shaking up gently ₂incubator is cultivated.

(3) Fugene HP transfection procedure

1) 12-24 hour before transfection, get the good and cell density of one bottle of upgrowth situation about 80% cell, after tryptic digestion, what add proper amount of fresh does not contain any antibiotic 10%FBS Growth of Cells nutrient solution, with suction pipe cell dispelled completely and form uniform cell suspension, in 24 orifice plates, every hole adds the cell suspension of 500 μ L, rocks culture plate gently and shakes up cell and be placed on 37 DEG C, 5%CO ₂12h-24h is cultivated in cell culture incubator.

2), during transfection, the cell density in orifice plate should reach more than 80%.Getting 0.5 μ g plasmid (pGL3-myoz1-pro ~ pGL3-myoz1-Q5) adds in the OPTI-MEM solution of 50 μ L, and piping and druming adds the Fugene HP of 1.5 μ L again after mixing gently, and room temperature leaves standstill 25min;

3) discard the cell culture fluid in orifice plate, with 1 × PBS or serum-free without after twice, dual anti-nutrient solution cleaning cell, add fresh cell culture fluid;

4) the 24 every holes of orifice plate add in (2) after mixed solution, and after the sway,postural culture plate of right-angled intersection all around mixes, put into 37 DEG C, 5%CO2 cell culture incubator cultivates 48h.

Embodiment 4: the detection of the two Luciferase activity of promotor TTR-pro of liver specific expression's gene TTR

The present embodiment arranges negative control plasmids pGL3-basic, positive control plasmid pGL3-control, with Renilla luciferase reporter gene pRL-TK(carrier purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega company) do internal reference plasmid (in order to correct transfection efficiency), collecting cell lysate after transfectional cell 48h, utilize luciferase reporter gene to detect lifetime measurement system to analyze the activity of the promotor TTR-pro of pig liver different expression gene TTR and tissue specificity, determine its active and liver organization expression specificity.Result shows (as shown in Figure 5): only pGL3-TTR-pro exists comparatively significantly transcriptional activity at Hepa1-6 cell, is about 15 times of negative control, its almost non-activity in 3T3-L1 and C2C12 cell.Result shows that TTR-pro promotor possesses the expression specificity that the function independently starting reporter gene expression also remains muscle tissue simultaneously

Embodiment 5:TTR-pro promoters driven external source BGL1 gene is in the checking of Hepa1-6 cells

The present embodiment take TTR-pro as research object, think that it has muscle specific and higher promoter activity concurrently, connect and eliminate the BGL1 gene C DS region of signal peptide, under the signal peptide effect of TTR-pro promotor and TTR gene, detect its expression in Hepa1-6 cell.

(1) be skeleton carrier with pGL3-Control Vector plasmid (purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, Figure 11 is shown in by its collection of illustrative plates), amplification pig BGL1CDS region.5 ' end of upstream primer adds Hind III digestion site and CCC protects base; downstream primer 3 ' holds interpolation 6 × his histone sequence label (being made up of six HHHHHH amino acid) sequence: CACCACCATCACCATCAT (having the base of two line in table 5) and terminator codon TAG; and add Xba I restriction enzyme site and CTAG protection base at 5 ' end of primer; by primer called after 1. BGL1:Hind III F and 1. BGL1His:Xba I R; primer sequence is in table 5; have single line place to be restriction enzyme site under base, pcr amplification parameter is with shown in table 2.

The design of table 5BGL1 gene clone primer

(2) restriction enzyme Hind III and Xba I double digestion pGL3-control, simultaneously enzyme cuts back to close the BGL1PCR product after purifying, and enzyme cuts system in table 6:

Table 6 enzyme cuts system

(3) be connected with BGL1CDS sequence by the pGL3-control carrier that above-mentioned enzyme cuts, 16 DEG C of connections are spent the night, and reaction system is in table 7.

Table 7 linked system

16 DEG C of water-baths are spent the night connection, proceeded in bacillus coli DH 5 alpha, screening positive monoclonal containing on the LB flat board of penbritin (Amp), PCR being carried out to recon and enzyme cuts qualification, positive recombinant being chosen and send the prosperous bio tech ltd order-checking of Beijing AudioCodes.For the correct positive colony that checks order in original bacteria liquid: the ratio of culture volume ratio=1:100 carries out enlarged culturing, with the plasmid that the can be used for cell transfecting extraction agent box (D6950-01 in a small amount that OMEGA company produces after 37 DEG C of shaking tables shake 16h, operate by the specification sheets of this test kit) extracting plasmid, called after pBGL1his-control.Identify the plasmid of extracting with Hind III and Xba I double digestion, it is described above that enzyme cuts system, and now plasmid only need add 1 μ L, and enzyme is cut and be the results are shown in Figure 7, is placed in-20 DEG C of refrigerators for subsequent use.

(4) with the genomic dna of Large White for touching plate (extracting genome DNA is for ordinary method); design primer is as table 8; amplification TTR gene promoter and signal peptide sequence; be TTRs by this sequence designations; wherein upstream primer adds Xho I restriction enzyme site and CCG protection base; downstream primer adds Hind III digestion site and CCC protects base, and base underscore place is restriction enzyme site, and pcr amplification parameter is with shown in table 2.

The amplification of table 8 TTR promoter-signal sequence

(5) restriction enzyme Hind III and Xho I double digestion pBGLhis-control, simultaneously enzyme cuts back to close the TTRs PCR primer after purifying, and enzyme cuts system in table 9

Table 9 enzyme cuts system

(6) be connected with TTRs sequence by the pBGLhis-control carrier that above-mentioned enzyme cuts, 16 DEG C of connections are spent the night, and reaction system is in table 10.

Table 10 linked system

16 DEG C of water-baths are spent the night connection, proceeded in bacillus coli DH 5 alpha, screening positive monoclonal containing on the LB flat board of penbritin (Amp), PCR being carried out to recon and enzyme cuts qualification, positive recombinant being chosen and send the prosperous bio tech ltd order-checking of Beijing AudioCodes.For the correct positive colony that checks order in original bacteria liquid: the ratio of culture volume ratio=1:100 carries out enlarged culturing, with the plasmid that the can be used for cell transfecting extraction agent box (D6950-01 in a small amount that OMEGA company produces after 37 DEG C of shaking tables shake 16h, operate by the specification sheets of this test kit) extracting plasmid, called after pTTRs-BGL1his-control.Identify the plasmid of extracting with Hind III and Xho I double digestion, it is described above that enzyme cuts system, and now plasmid only need add 1 μ L, and enzyme is cut and be the results are shown in Figure 9, is placed in-20 DEG C of refrigerators for subsequent use.

(7) the plasmid called after pTTRs-BGLhis-contorl will built, transfection Hepa1-6 cell, each plasmid does 3 repetitions, and the Hepa1-6 cell simultaneously arranging 3 any plasmids of untransfected is blank.48h after transfection, the RNA carrying out Hepa1-6 cell with the RNA extraction test kit of OMEGA company extracts (operating by test kit specification sheets), carries out reverse transcription (operating by the specification sheets of the Reverse Transcription box of Thermo company), the expression of BGL1 in Hepa1-6 cell is detected by RT-PCR, BGL1 and reference gene primer are in table 11, and reaction system and program are as following table 12.

Table 11 increases the design of primers of Hepa1-6 cell cDNA

Table 12 RT-PCR amplification system

Result shows, in transfection in the Hepa1-6 cell of recombinant plasmid pTTRs-BGLhis-contorl, the expression of BGL1 can be detected, and do not have not express in the cell of transfection recombinant plasmid, prove that liver-specific promoter TTR-pro can drive external source BGL1 at the Hepa1-6 cells of mouse really.

Claims (2)

1. the promotor that the liver organization of a regulation and control pig liver tissue gene TTR is specific expressed, its nucleotide sequence is as shown in sequence table SEQ ID NO:3.

2. promotor according to claim 1 is in the application of regulatory gene in pig liver tissue specifically expressing.